Two mechanisms of cell death in the body are recognized, necrosis and apoptosis. Apoptosis is the process of programmed cell death, described by Kerr et al in 1992 [Kerr JFR, Wyllie AH, Currie AR (1992). “Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. “British Journal of Cancer 26: 239–257”], by which steady-state levels of the various organ systems and tissues in the body are maintained as continuous cell division and differentiation takes place. Cells undergoing apoptosis often exhibit distinctive morphological changes such as a pronounced decrease in cell volume, modification of the cytoskeletons resulting in pronounced membrane blebbing, a condensation of the chromatin, and degradation of the DNA into oligonucleosomal fragments. Following these morphological changes, an apoptotic cell may break up into a number of small fragments known as apoptotic bodies, consisting essentially of membrane-bound bodies containing intact organelles, chromatin, etc. Apoptotic bodies are normally rapidly removed from the body by phagocytosis by macrophages, dendritic cells and other antigen presenting cells, before they can become lysed and release their potentially pro-inflammatory intracellular contents.
In simple outline, apoptosis is thought to proceed as follows. Three phases can be identified in the apoptotic mechanism of programmed cell death:
Induction phase;
Effector phase; and
Degradation phase.
The induction phase is dependent, in part, on specific interactions of death-inducing signals at the cell surface membrane. One common signal is initiated by the binding of specific ligands to receptors of the TNF receptor family present on the cell membrane. One important such receptor is Fas (APO-1, CD95), which interacts with Fas-ligand to initiate apoptosis.
The effector phase, activated by the binding of receptors and ligands of the induction phase, leads to the activation of caspases, cystinyl-aspartate-requiring proteinases (proteolytic enzymes), including caspases 1 and 8. This activation may be associated with a change in the permeability of mitochondria, allowing the release of cytochrome-c which is involved in caspase activation. Activated caspases initiate a chain of lethal proteolytic events culminating in the changes in chromatin and cytoskeletal components seen in apoptosis.
Many cells undergoing apoptosis can be identified by a characteristic ‘laddering’ of DNA seen on agarose gel electrophoresis, resulting from cleavage of DNA into a series of fragments. These changes occur a few hours before death of the cell as defined by the ability of a cell to exclude vital dyes. The appearance of DNA laddering on agarose gel electrophoresis following extraction of DNA from cells is one recognised method of identification of apoptosis in cells [Loo, D. T. and Rillema, J. R. (1998) “Measurement of Cell Death,” Methods in Cell Biology 57: 25 1–264], although it is not always sensitive enough to detect apoptosis. In situ labelling of nuclear DNA fragmentation, for example, using commercially available terminal dUTP nick end labelling (TUNEL) assays, is an alternative and more reproducible measure for the determination of fragmented DNA in apoptotic cells and cells undergoing apoptosis [Gavrieli Y, Sherman Y, Ben-Sasson S A (1992)”, “Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation”. Journal of Cell Biology 119: 493–501].
During apoptosis, phosphatidylserine becomes exposed externally on the cell membrane [Fadok V A, Voelker D R, Campbell P A, Cohen J J, Bratton D L, Henson P M (1992), “Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages”. Journal of Immunology 148: 2207–2216] and this exposed phosphatidylserine binds to specific receptors to mediate the uptake and clearance of apoptotic cells in mammals [Fadok V A, Bratton D L, Rose D M, Pearson A, Ezekewitz R A B, Henson P M (2000), “A receptor for phosphatidylserine-specific clearance of apoptotic cells”, Nature 405: 85–90]. The surface expression of phosphatidylserine on cells is another recognised method of identification of apoptotic cells.
Changes in mitochondrial integrity are intimately associated with apoptosis, resulting in alterations in mitochondrial membrane permeability and the release of cytochrome-c from the mitochondria into the cell cytoplasm [Susin, S. A., Lorenzo, H. K., Zamzami, N., Marzo, I, Brenner, C., Larochette, N., Prevost, M. C., Alzari, P. M. and Kroemer, G. (1999) “Mitochondrial Release of Caspase-2 and -9 during the Apoptotic Process”, Journal of Experimental Medicine, 189: 381–394]. Measurement of changes in mitochondrial membrane potential, reflecting changes in mitochondrial membrane permeability, is another recognised method of identification of apoptotic cells.
A number of other methods of identification of cells undergoing apoptosis and of apoptotic cells, many using monoclonal antibodies against specific markers for apoptotic cells, have also been described in the scientific literature.
Necrosis, in contrast, is cell death of a pathological nature, resulting from injury, bacterial toxin effects, inflammatory mediators, etc., and involving membrane rupture and release of intracellular contents to the surrounding tissue, often with harmful inflammatory consequences. Accordingly, one of the ways in which necrotic cells may be detected and characterized is by detection of compromised cell membranes, e.g. by methods of staining with propidium iodide followed by flow cytometry or microscopy.